There have been several approaches to gene therapy, each of which has inherent drawbacks. For example, recombinant retroviral vectors have been used to integrate a gene of interest into a target cell genome. However, retroviruses integrate efficiently only into replicating calls and they are difficult to concentrate and purify. Further, there is concern that retroviruses are carcinogenic.
Several DNA viruses, such as adenovirus, have also been engineered to serve as vectors for gene transfer. But adenoviruses can carry only a limited insert and are often restricted in the range of cells they infect. Moreover, adenoviruses fail to integrate their inserts into the host genome leading to only transient expression. Host immunity also pose problems for repeated administration. A further difficulty is that recipient cells generally express viral proteins in addition to the therapeutic gene, and these viral proteins cause immune responses and subsequent inflammation in the recipient organ.
Some of these drawbacks are overcome by utilizing adenoassociated virus (AAV), which is a single-stranded DNA parvovirus. AAV is a defective virus that productively infects only cells in which certain functions are provided by a co-infecting helper virus such as adenovirus and herpesvirus. Infection of cells with AAV in the absence of helper functions results in integration of AAV into the host call genome without replication. The AAV genome has two copies of a 145-nucleotide-long ITR (inverted terminal repeat), one at each end (Srivastava et al., J. Virol., 45, 555-564 (1983)). The ITR sequences provide an origin of replication and also mediate integration and excision of the AAV genome from the cell genome.
The sequence between the ITRs of about 4470 nucleotides contains two open-reading frames for rep and cap genes (Hermonat et al., Virology 51, 329-339 (1984)). The cap gene encodes capsid proteins. The rep gene encodes proteins known to be required for replication. A possible additional function of rep proteins, integration of AAV DNA into the host genome, remains controversial. There is some evidence that rep.sup.- vectors show reduced preference for site-specific integration into chromosome 19. However, it has been reported that the overall integration frequency of rep.sup.- vectors is higher than that of comparable rep.sup.+ vectors. McLaughlin et al., J. Virol. 62, 1963-1973 (1988).
AAV is nontransforming and not associated with any disease (Ostrove et al., Virology 113, 521 (1981); Cukor et al., in The Parvoviruses (ed. Berns, Plenum, N.Y., 1984)). Further, AAV virions are resistant to physical treatments, such as sonication and heat inactivation not tolerated by other viruses during purification (Samulski et al., J. Virol. 63, 3822-3828 (1989)). Like retroviruses, AAV integrates into the host cell genome upon infection (Kotin et al., Proc. Natl. Acad. Sci. USA 87, 2211-2215 (1990); Samulski et al., EMBO J. 10, 3941-3950 (1991)). However, unlike retroviruses, AAV preferentially integrates at a specific chromosomal site (19q13.3) (AAVI). At this site, AAV does not cause any significant alteration in the growth properties or morphological characteristics of human cells. Furthermore, integration of AAV into the cellular genome can occur in nonproliferating cells. (Lebkowski et al., Mol. Cell. Biol. 8, 3988-3996 (1988)).
Nevertheless, existing methods of using AAV for gene transfer have several drawbacks. A major problem limiting the practical use of recombinant AAV is that AAV production methods are inefficient and laborious (Lebkowski et al., 1988, supra; Samulski et al., 1989, supra; Muzyczka, Curr. Top. Microbiol. Immunol. 158, 97-129 (1992)). In recombinant AAV, all protein coding sequence (such as cap, and rep) are usually replaced by the exogenous gene of interest. Recombinant AAV is replicated by co-transfecting a cell bearing the AAV vector carrying the gene of interest, together with a helper AAV plasmid that expresses all of the essential AAV genes, into adenovirus-infected cells, which supply additional helper functions necessary for AAV replication and the production of new viral particles. Using this approach, it is difficult to obtain the high yields of packaged viral genomes that are required for use in gene therapy. Further, the preparation of recombinant AAV may be contaminated with wildtype AAV from the helper plasmid or infectious virions of the helper virus, such as herpes or adenovirus. An additional drawback from packaging AAV genomes before introduction of cells is that the maximum size of an insert compatible with packaging is limited to about 5 kb.
Some of the disadvantages stemming from the use of viral vectors are avoided by transfecting a DNA fragment into cells nonbiologically, for example, by lipofection, chemical transformation or electroporation. In this approach, ample amounts of pure DNA can be prepared for transfections, and much larger fragments can be accommodated. To-date, however, such approaches have been limited to cells that can be temporarily removed from the body. Furthermore, the efficiency of gene integration has been very low, about one integration event per 1,000 to 100,000 cells, and expression of transfected genes has been limited to days in proliferating cells or weeks in nonproliferating cells. Without integration, expression of the transfected gene may be limited to several days in proliferating cells or several weeks in nonproliferating cells due to the degradation of the unintegrated DNA.
A further method has been proposed in which a recombinant vector containing AAV ITR sequences but lacking all other AAV sequences is surrounded by cationic lipids and introduced into a cell by lipofection. Philip et al., WO 95/07995. However, this method does not result in efficient integration. Lebkowski et al., Society Francaise de Microbiologie, VIth Parvovirus Workshop, Abstract S5 #7.
Accordingly, there is a need for improved AAV cloning vectors and methods of transferring the same into recipient cells. The present invention fulfills this and other needs.